Our objective is to elucidate the role of the interferon (IFN)-induced protein kinase in the actions which cloned and natural IFNs mediate on viral and host functions.
The specific aims are: (1) To attempt to further purify the IFN-induced protein kinase which catalyzes the phosphorylation of ribosome-associated protein P1 and the Alpha subunit of protein synthesis initiation factor eIF-2; the kinase activity copurifies with protein P1. Starting material may include cells grown in culture, animal tissue or host cells within which P1 cDNA is expressed. The purification scheme presently used may be modified to include affinity chromatography and/or preparative HPLC steps. We plan to continue our biochemical characterization of P1 preparations with emphasis on the following: the study of the possible role of mRNA secondary structure in kinase activation and translation inhibition; the examination of the mechanism and role of the inhibition of P1/eIF-2Alpha kinase activity by adenovirus VAI RNA; and the examination of the arrangement of P2 on the 4OS ribosomal subunit. (2) To attempt to construct and characterize a full-length cDNA copy of the mRNA endocing human protein P1. Protein P1 cDNA clones will be utilized in studies on the regulation of P1 expression in interferon-treated and virus-infected animal cells. We plan to attempt to obtain expressin of P1 in prokaryotic and eukaryotic cells after insertion of P1 cDNA into appropriate vectors as an approach to structural and functional analyses of P1. (3) To continue our analysis of the phosphorylation status of protein P1 and initiation factor eIF-2Alpha in intact cells in culture in the presence and absence of treatment with interferon and infectin with himan reovirus or adenovirus. Wild-type reovirus is IFN sensitive and activates the P1/eIF-2Alpha kinase, whereas wild-type adenovirus is relatively IFN-resistant and inhibits the kinase. These serotypes of reovirus will be examined which differ appreciably in their capacity to inhibit cellular protein synthesis, and wild type andd mutant strains of adenovirus will be studied which differ in their ability to produce VA RNA and synthesize late viral proteins. The effect of IFN type (Alpha, Gamma) and cell type (epithelial, fibroblast) will be examined. Two dimensional electrophoresis and immunoblot procedures will be used to quantitate P1 and eIF-2Alpha phosphorylation.
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